Factors affecting Schwann cell basal lamina formation in cultures of dorsal root ganglia from mice with muscular dystrophy

Developmental Brain Research, 6 (1983) 57-67 Elsevier Biomedical Press

57

Factors Affecting Schwann Cell Basal Lamina Formation in Cultures of Dorsal Root Ganglia from Mice with Muscular Dystrophy C. J. CORNBROOKS, F. MITHEN, J. M. COCHRAN and R. P. BUNGE

Department of Anatomy and Neurobiology, and (F.M.) Department of Neurology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110, and (J.M.C.) Department of Anatomy, Temple University, 3420 N. Broad Street, Philadelphia, PA 19140 (U.S.A.) (Accepted April 22nd, 1982)

Key words: murine muscular dystrophy - - neurogenic defects - - neuron - - Schwann cell - - basal lamina - - ensheathment

Pure populations of sensory neurons (N), Schwann cells (S) and fibroblasts (Fb) were established in culture from normal and dystrophic (dy) mice in order to investigate the cellular origin(s) of the peripheral nervous system abnormalities present in murine muscular dystrophy. These cell types were placed together in various combinations and their subsequent interactions were monitored with the light and electron microscope. The formation of the basal lamina (BL) which in normal tissue, completely surrounds the external aspect of the Schwann cell (when in contact with axons) was documented by morphometric analysis of electron micrographs. Defects in Schwann cell BL formation, observed throughout the PNS of the dy mouse in vivo, were used as a marker for the expression of the dystrophic abnormality in culture. Initially mature cultures of dy tissues containing only S and N (SN) without Fb were examined and found tp contain an incomplete BL that surrounded only 82.8 ± 12.2~o of the externally directed plasmalemma of axon-related Schwann cells. The following recombination cultures were established: (1) normal S were placed on dystrophic N ; (2) dystrophic S were placed on dystrophic N ; (3) dystrophic S were placed on normal N; and (4) normal Fb were added to a dystrophic SN culture. After a 5-week period, the BL formed by normal S in direct contact with dystrophic N was thick and continuous (97.7 -:-'2.2 coverage). On the other hand, in culture situations (without Fb) containing dystrophic S in contact with either dystrophic or normal neurites, the BL coverage was considerably less (58.5 -t- 14.8~ and 55.4 ± 13.2~o, respectively). The addition of normal Fb obtained from sciatic nerve explants to dystrophic SN cultures in time resulted in the formation of a morphologically complete BL (98.9 ± 1.4 ~. coverage). We conclude that neuronal signal(s) are adequate to induce complete BL formation by Schwann cells in the dystrophic tissue but that dystrophic Schwann cells are incapable of forming a complete BL. Furthermore, this deficiency of dy Schwann cells is apparently corrected by the presence of normal Fb by an unknown mechanism. INTRODUCTION D u r i n g n o r m a l development of the peripheral nervous system (PNS), the S c h w a n n cell (S) interacts with the n e u r o n (N) to provide e n s h e a t h m e n t or m y e l i n a t i o n o f axonal processes 46. D u r i n g the early period after the initial appearance o f S c h w a n n cells, their p o p u l a t i o n is substantially e x p a n d e d ; tissue culture experiments suggest this expansion is controlled by a mitogen present o n the axonal surface 29,39-41,49. S u b s e q u e n t l y smaller axons are ensheathed a n d larger axons myelinated by S c h w a n n cells; the signal d e t e r m i n i n g which S c h w a n n cells ensheath a n d which progress to m y e l i n a t i o n is k n o w n from in vivo t r a n s p l a n t a t i o n work to also originate from the axon 1,47. U p o n the establishment 0165-3806/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press

o f a long-lasting relationship between the n e u r o n a l process a n d the S c h w a n n cell, a basal l a m i n a (BL) is formed2,11, 45. With time this layer of extracellular material completely envelops the external aspect of the S c h w a n n cell p l a s m a l e m m a , i.e. that p o r t i o n n o t related directly to axons. F r o m tissue culture experiments, it has been shown that the S c h w a n n cell is capable of secreting a morphologically complete BL when grown in c o n t i n u o u s contact with n e u r o n s with or without fibroblasts (Fb) 11. The biochemistry o f the S c h w a n n cell BL has n o t been a subject of rigorous investigation b u t it is k n o w n to be partially sensitive to collagenase 11 a n d rendered invisible to electron microscopic visualization after trypsin t r e a t m e n t 11. The BL formed by other m a m m a l i a n cells has been shown to be composed o f a complex

58 arrangement of molecules including glycosaminoglycans and proteoglycans in addition to collagen and other proteinsG,~-",2z,"6,37:L The mechanism by which these and other components are combined to form a functional matrix has not been elucidated. The dystrophic mouse is known to express neurological abnormalities of unknown cellular origin(s). Severe lesions in the peripheral nervous system are characterized by a deficient ensheathment of axons by Schwann cells 7. Amyelinated areas have been demonstrated in the cranial nerves and the dorsal and ventral roots at certain vertebral levels3:. 7,8. Subsequent examination of the more distal regions of the dystrophic PNS revealed subtle lesions including increased lengths of the nodes of Ranvier 9, 3'~, decreased Schwann cell proliferation in the spinal roots ~°,24, and a patchy or deficient BLe8, a3,a4. In initial tissue culture experiments by Okada et al. ~2, organotypic explants from the dystrophic dorsal root ganglia ( D R G ) expressed only the less severe abnormalities previously described in vivo. Neither the in vivo nor tissue culture studies have clearly identified the cell type(s) responsible for the abnormalities. Methods are presently available in tissue culture which permit the isolation of pure populations of PNS neurons and Schwann cells from both normal and dystrophic mice 48. It is therefore possible to mix pure populations of each cell type from the normal and abnormal animals and monitor their subsequent interactions. Light and electron microscopic methods were used to observe the maturation of the Schwann cell-neuron (SN) relationship as assessed by the ensheathment of axons with particular emphasis on the formation of the BL. We have employed this scheme to determine that the dystrophic Schwann cell is responsible for the morphological abnormalities in the BL which forms in culture. We have also been able to correct the BL defects expressed in culture by the addition of normal mouse tibroblasts to Schwann cell-nerve cultures established from dystrophic tissues. Preliminary reports of these studies have been presented15, 2°. MATERIALS AND METHODS

Culture methods" Homozygous mice, either C57BL/6J

-~/-+- or

C57BL/6J dy"-J/dy"J were obtained from Jackson Laboratories (Bar Harbor, Maine) and bred in facilities in our labolatory. Typically, 18-day fetal mice were surgically removed and stored under sterile conditions in Lcibowitz-15 nredium. The fetuses were killed by decapitation and after removal of the spinal cord, only the lumbar and sacral D R G were dissected from the vertebral column. The connective tissue sheath of each ganglion was removed by microdissection and 5-7 ganglia were placed in each 24 mm Aclar minidish:. These Aclar dishes had been previously coated with one layer of reconstituted, ammoniated, rat tail collagen .5, and covered with 4 drops of medium. Cultures were maintained in closed, humidified jars containing 5 '),, CO2-95 ~,~ air at approximately 34 °C. Explant cultures were prepared by a modification of the method of Salzer and Bunge :~9. For 4 days the medium 1 contained 65 oJ /o Eagle's minimal essential medium with Earle's salts (MEMES), 25 vol. ~ human placental serum, 10vol. ~,, 9-day chick embryo extract, 2 mM L-glutamine, 600 mg°/o glucose, and 5-bromo-2'-dcoxyuridine at 50 pg/ml and 10-20 U of nerve growth factor (NGF). This feed was changed on day 2. On day 4 the cultures were submitted to 10 min of ultraviolet irradiation from a General Electric germicidal lamp (C30T8) at the distance of 6 in. and the feed was changed to medium 11 which contained 75 vol. ",i MEMES, 10 vol. '!~,human placental serum, 2 vol. ",, 9-day chick embryo extract, 2 mM L-glutamine, 600 m g % glucose, N G F , uridine (2 :,:: 10 -..5 M), 5-fluorodeoxyuridine (2 × 10 ~ M) and cytosine arabinoside (2 × 10 -5 M). On day 6 the cxplant areas containing primarily the neuronal somata were excised and transplanted to a second Aclar dish. Cultures grown to contain Schwann cells and neurons (SN) were subsequently maintained in medium IV, which is identical to medium I except the 5-bromo-2'-deoxyuridine was omitted. Cultures grown to contain neurons only (N) were recycled on the aforementioned antimitotic treatment and then maintained for one month, post-transplant on medium II1, which is the same as medium 1I except the cytosine arabinoside is omitted. After the antimitotic treatment, 4 drops of medium were replaced 3 times weekly. After 1 month, N cultures were switched to medium 1V and monitored by light microscopy for the appearance of non-neuronal cells. N cultures

59 were used as hosts to receive transplants of Schwann cells from the outgrowth areas of SN donor cultures. Fibroblasts were obtained from the outgrowth zone of small fragments of neonatal normal mouse sciatic nerve which had been maintained in culture for several weeks in medium IV. After one month post-transplant in medium IV, areas approximately 1 mm z in the SN cultures containing neurites and Schwann cells on the collagen matrix were removed by microdissection and transferred with the collagen matrix to host N cultures (Fig. 1). The donor transplants were placed such that the donor's Schwann cells were in direct contact with host's neurites sandwiched between the two layers of the collagen matrix. To aid in the attachment of the transplant, the medium volume was lowered overnight. These recombination cultures were maintained in 4 drops of medium IV for 5 weeks and the growth and phenotypic expression were monitored by light microscopy prior to fixation for electron microscopic observations. Similarly, normal Fb from sciatic nerve cultures were transferred to dystrophic SN cultures. The cell recombination cultures included the following groups: (1) dystrophic Schwann cells placed on normal neurons [S(dy) X N ( ÷ ) ] ; (2) dystrophic Schwann cells placed on dystrophic neurons [S(dy) x N(dy)] ; (3) normal Schwann cells placed on dystrophic neurons [S(-~-) x N(dy)]; and (4) normal fibroblasts placed on dystrophic Schwann cell-neuron cultures [Fb(q-) x

wise in ethanol and embedded in low viscosity resin. Regions of the embedded block containing areas of tissue were excised and mounted on a plastic capsule for sectioning. The blocks were sectioned perpendicular to the plane of the culture dish on a PorterBlum MT-2 ultramicrotome equipped with a diamond knife (Dupont). Silver-gold sections were stained with lead citrate and examined with a Philips 300 transmission electron microscope.

Morphometric analysis Electron micrographs from all categories of culture preparations were randomly mixed and covered with a clear sheet of cellulose acetate. Basal laminae which were associated with axons ensheathed by Schwann cells were marked by pen such that thin or interrupted segments were delineated. The percent

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Electron microscopy Cultures were rinsed for 30 min in Earle's balanced salt solution (Gibco) at 37 °C and fixed overnight at 4 °C in modified Karnovsky's fixative consisting of 5 % glutaraldehyde (EM Sciences), 4 % paraformaldehyde, 0.3% sodium chloride, 2 mM calcium chloride and 1% dimethylsulfoxide in 0.08 M sodium cacodylate buffer (pH 7.4). Cultures were rinsed in phosphate-buffered saline (pH 7.4) for 30 min and postfixed in 1% osmium tetroxide with 1.5 % potassium ferrocyanide in 0.1 M potassium phosphate buffer, (pH 7.2) at 4 °C. Following a 30 min rinse in 0.1 M maleate buffer (pH 4.7), the cultures were stained en bloc in 1% uranyl acetate in 0.1 M maleate buffer (pH 4.7) for 24 h at room temperature. The tissue was then dehydrated step-

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Fig. I. A schematic representation of the method used to transfer Schwann cells from a donor SN culture to a host N culture. Explant cultures are established from rat dorsal root ganglia and treated (see Materials and Methods) in such a manner to allow the growth of neurons (A) or both neurons and Schwann cells (B). Fibroblasts are eliminated in both types of cultures. After I month in culture, a rich arborization of neurites grew in a distal direction from the explants (solid arrows). Depending on the media used, these neurites may be populated by S (open arrows). Selected areas from the donor SN cultures are surgically removed and transplanted onto the neuritic region of the host, N culture (C). After a short period of time, S which are placed in direct contact with the host neurites begin to proliferate and repopulate the neurites. (D). S and N were allowed to relate to each other for at least a 5week period and subsequently examined at the LM and EM levels.

60

Fig. 2. A light micrograph of a living recombination culture schematized in Fig. IC and D. Tissue from a SN donor culture which contains S (arrow) has been placed on the distal portions of the host neurites (A). Within an 18-h period, exogenous, bipolar S (arrows) have begun to proliferate in response to the axolcmmal bound mitogen (B). Subsequently, the Shavc left the immediatc implant region, migrate in direct contact with the ncuritcs and will eventually repopulatc the naked neurites. (From a culture preparation grown by Dr. Patrick Wood.) (Magnitication: 46 ...)

coverage was determined by tracing these lines on a magnetostrictive digitizing tablet (Houston Instrument, Austin, TX) interfaced to a microcomputer (Ithaca Intersystems, Ithaca, NY) programmed for morphometric analysis. RESULTS The procedures outlined above provide explant cultures from the normal and dystrophic mouse D R G grown without the presence of Fb. Living cultures from both types of mice maintained comparable neuritic growth rates and patterns throughout their 2 or more months tenure in the culture chamber. Neuronal somata extended neurites which often traversed a distance of greater than 5 mm after 1 month in culture. In general, in cultures maintained in medium IV, the greater majority of the naked neurites were populated with Schwann cells; those maintained in medium III contained neuronal

somata and outgrowing ncurites only. It was then possible to transfer Schwann cells from donor SN cultures to bare neurites of host N cultures (Fig. 1). The success of these recombination cultures dependcd on the placement of the Schwann cells in dircct contact with the host neurites. Within a short period of time after they were correctly positioned, the donor Schwann cells began to proliferate and migrate in direct contact (only) with the host neurites in both proximal and distal directions (,Fig. 2). Within 4-5 weeks, the exogenous Schwann cells had populated the major portions of the host neurites which were initially contacted by the cellular implant. There was no apparcnt difference in the proliferative rate of the S in any of the recombination cultures, although quantitative studies addressing this question were not done. Typically, these Schwann cells occupied an area on only one side of the cxplant and were not witnessed to migrate through the explant region which contained the neuronal somata. Con-

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Fig. 3. Representative electron micrographs from 3 types of cultures which were fixed 5 weeks after exogenous S were transferred onto the host neurites (A-C) and a SN culture from a dystrophic animal fixed 5 weeks after the cessation of the antimitotic treatment used to suppress Fb formation (D). Incomplete BL coverage (arrows) outlines the outer, dystrophic S plasmalemma in both the S(dy) x N(x-) (A) and the S(dy) × N(dy) (B) recombination cultures. Numerous unensheathed axons (stars) were also prominent entities in both types of cultures containing dystrophic S. The BL formed by the normal S when in direct contact with dystrophic neurites (C) in the S(-~.) × N(dy) cultures was thick and continuous. Ensheathment of numerous neurites (stars) and the BL coverage (arrows) was also abnormal in SN cultures (D) derived from the dystrophic animals. (Magnification for A, B, D: 55,000x ; for C:44,000 x . )

62 "FABLE I Basal lamina coverage by normal o1" dystrophic Schwann cells

BL coverage: portion of the Schwann cell plasmalemma (apposed to the extracellular matrix) covered by BL i. standard deviation. Number of cultures: cultures from which the samples were measured. BL lengths: number of BL lengths measured. Actual BL length: actual length (nm) measured. Culture

% BL coverage

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Actual B L length nm ,, 10~

S(:-) x N(dy) SN(dy) S(dy) x N(-!-) S(dy) :~: N(dy) Fb(+) × SN(dy)

97.7 --- 2.2 82.8 5:12.2 55.4 ___ 13.2 58.5 5_ 14.8 98.9 ± 1.4

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48 22 23 13 42

1.59 0.92 0.56 0.40 2.45

sequently, neurites on the opposite side of the explant remained unpopulated by non-neuronal cells. Periodic monitoring of explants which received transplants was necessary to detect the presence of Schwann cells endogenous to the host explant. Occasionally, Schwann cells resident in the explant are capable of surviving the 1 month of antimitotic treatment. These endogenous Schwann cells initially appear in the neuritic display immediately adjacent to the neuronal somata. They are distributed around the entire circumference of the explant and proliferate in the direction distal to the explant. Electron microscopic methods were used to investigate the ability of normal and dystrophic S to form BL. Samples in the SN cultures were taken from an area approximately 1 mm from the explant where S had been in contact with neurites for the longest period of time. In mature (at least 4 weeks in culture) SN cultures from normal mice, the BL was a continuous envelope which surrounded the S domain with a characteristic thickness of approximately 30 nm 3'. In contrast, the BL in SN cultures prepared from dystrophic mice remained thin or absent in some areas (Fig. 3D). BL covered only 82.8 :!- 12.2~,~ (Table 1) of the S plasmalemma apposed to the extracellular matrix. This phenomenon was observed in dystrophic tissue maintained for 2 months in culture. In addition, this extracellular matrix component was not present where the S was in close apposition to an unensheathed neurite or another S. In the recombination cultures, both the normal and dystrophic Schwann cells remained in contact with and proliferated to populate either type of host

neurite. In all cases, these cells repopulated a substantial portion of the previously naked neurites within the 5-week time period studied. There was no apparent difference in proliferative rate of the Schwann cells in any of the culture situations examined. Electron microscopic observations of the S( i ) ;, N(dy) cultures revealed a continuous BL of normal thickness surrounding 97.7 --iz 2.2 ~ (Table i) of the Schwann cell plasmalemma (Fig. 3C). Schwann cells obtained from the dystrophic SN donor expressed a patchy, discontinuous BL when they were in contact with neurites grown from dystrophic (Fig. 3A) or normal animals (Fig. 3B). Only 55.4 -b 13.2~,~ or 58.5 .... 14.8~ (Table I). respectively of the S plasmalemma was associated with BL material. In addition, dystrophic Schwann cells demonstrated an inability to successfully ensheath (i.e. to provide complete cytoplasmic covering) the neurites of either the dystrophic or normal neurons (Fig. 3A, B). Thus, under culture conditions, a thick, continuous BL was only found around normal S and a thin, discontinuous BL was consistently found around dystrophic S regardless of the genotype of the host neurite. In one S(-t-) × N(dy) culture, the transplant of normal Schwann cells had been placed on the neuritic outgrowth of a host dystrophic explant and these Schwann cells began to proliferate and inhabit the neu~ite domains. Concomitantly, endogenous Schwann cells appeared in a second explant in the same dish and began to repopulate the neurites. In no case were Schwann cells grown in serum-supplemented media observed to migrate without sustaining direct contact with neurites. The fact that the neuritic arborizations of the two explants were not

Fig. 4. An electron micrograph from a F b ( + ) x SN(dy) culture. Fb from a normal sciatic nerve preparation were added to SN(dy) cultures and maintained in medium IV for at least 4 weeks. Axons (stars) are well ensheathed and the Schwann cell-axon unit is circumscribed by a thick, continuous BL. (Magnification: 30,500 x .) Fig. 5. An electron micrograph from a F b ( + ) x SN(dy) culture demonstrating a morphologically complete BL (closed arrows), and plentiful endoneurial connective tissue typical for cultures containing Schwann cells, neurons, and fibroblasts; however, the axons contained in this fascicle (stars) were not individually ensheathed by Schwann cells as would occur in normal tissue maintained in culture for 4 weeks. (Magnification: 30,500 x .)

64 contiguous precluded the possibility that the exogenous normal S and the endogenous dystrophic S could change explants. Schwann cells on both explants were allowed to mature for 5 weeks and examined in electron micrographs. 3he endogenous dystrophic--SN culture developed the characteristic thin, patchy BL previously reported, ~.hereas the nerve-Schwann cell units of the S ( : ) :.: N(dy) explant exhibited a thick and continuous BL. Additional extracellular matrix components, the collagen fibrils, were examined in each culture situation. Typically, only the smaller (below 25 nm) diameter collagen fibrils were seen in the SN cultures t''. No difference in size or distribution of collagen fibrils was seen in SN cultures from the normal or dystrophic tissue. The fibrils were generally oriented parallel to the axis of elongation of the dystrophic or normal Schwann cell-neurite unit. After the establishment of dystrophic-SN cultures, it was possible to repopulate the cxplant with normal Fb obtained from explant cultures of normal mousc sciatic nerves. These exogenous Fb rapidly proliferated and eventually overgrew a substantial portion of the explant and its outgrowth region. The BE lbrmed around SN units in the regions o f t h c Fb transplant was continuous (98.9 ~:: 1.4')i, coverage, Table 1) and of normal thickness (Fig. 4). This observation contirmcd more extensive experiments of this type reported elsewhere ~6. Thus in the presence of normal tibroblasts, the BL deficiency of the dystrophic S is not expressed. However, an inability by the dystrophic S to cnsheath numerous naked axons was apparent (Fig. 5). DISCUSSION A Schwann cell which exists in the migratory stage in vivo, either early in development or as a result of injury, does not form a BL z,'-'~. Electron microscopic evidence suggests that the acquisition of BL is necessarily preceded by the establishment of a relationship with a neuronal process(es) ~,5. Prior to forming a permanent relationship, Schwann cell processes interpose between unenshcathed naked axons and subdivide them into nerve-Schwann cell units. This stage in turn leads to the ensheathment of the neuronal processes, cessation of Schwann cell proliferation, and the concomitant appearance of a

BL and collagen fibrils. In the early stages of the Schwann cell-axon relationship, the BL appear~ immature in that it is patchy and discontinuous 4~'. I11 the mature stage, the BE is a thick, unilbrm layer of extraccllular material that surrounds that portion of Schwann cell plasmalemma which faces outward, away from the enclosed axons. In tissue culture preparations the same sequence of events, from Schwann cell proliferation ~°,41,1:' to basal lamina formation ~.v" may be observed and has been sho~ n to depend on axon contact. The recent in vitro studies have resolved some of the earlier uncertainty over the ability of Schwann cells to form extracellular matrix components (such as the BL and collagen fibrils). Several laboratories have successfully shown the production of collagen and/or BE by clonal lines of Schwann cells, or Schwann cells grown as primary culturesl".l.% Both in vivo and tissue culture observations suggest that the tbrmation of the Schwann cell BL requires the interaction of at least two cell types, i.e. the neuron and the Schwann cell. Bunge et al. 1~ clearly demonstrated that neither cell type when grown as a pure population forms morphologically visible extracellular components. However, when these two cell types are allowed to contact each other, the BL and collagen fibrils develop. Matrix components and environmental factors also affect the ability of the Schwann cell to form its BL. Axonal contact is not sufficiently informative in itself to allow the Schwann cell to make extracellular components; apparently, contact with a suitable matrix is also requiredl:~.vL When groups of Schwann cells are deprived of contact with a collagen-containing substrate by their attachment to suspended neuritic fascicles, the S are not capable of normal ensheathment of axons. If forced into physical contact with a collagen matrix, these Schwann cells undertake normal axonal ensheathment and the formation of mature extracellular matrix. Humoral factors have also been shown to play a role in Schwann cell differentiation "~°,:~1. Media which contain no embryo extract or serum components are permissive for Schwann cell recognition of the axolemmal-bound mitogen: however, there is a virtual absence of ensheathment, myelination, and of BL and collagen fibril formation. The neurogenic component of murine dystrophy

65 was first recognized in nerve root regions by Bradley and Jenkison 7 and by Solafsky and Sterling 38. Additional observations by Madrid et al. 28 demonstrated a patchy deficiency of the basement membrane of myelin associated Schwann cells. The complete absence of the basement membrane in amyelinated zones of the nerve root has also been shown to be associated with 'uncommitted' cells which are likely to be Schwann cells a6. These cells have the characteristics of immature or traumatized Schwann cells in that they undergo mitosis even in the adult animal. The thorough studies of Jaros and Bradley 24 documented that the BL deficiency was associated with both types of ensheathed axons, myelinated and unmyelinated, throughout the PNS. One is struck by the similarity of the dystrophic deficiencies in axon ensheathment and BL formation and the observations of the immature stage prior to the establishment of a permanent Schwann cell-axon relationship. Indeed, Jaros and Bradley ~5 have documented at least 4 situations of atypical Schwann celi-axon relationships which are similar to features of CNS myelination. These anomalies were attributed to an immature, incomplete BL of the Schwann cell which in its absence is thought to permit Schwann cell mobility. The complex interplay of the cell types and humoral factors necessary for normal enshcathment and BL formation by the Schwann cell provide numerous candidates for the causative agent. The axon, known to be necessary for Schwann cell mitosis and myelinationL47,4~, may not provide the correct signal to the ensheathing cell. On the other hand, the Schwann cell may be incapable of reading the signal and proceeding with the normal metabolic duties of differentiation. A third possibility is that ensheathment and/or BL formation requires an extracellular component which is missing or abnormal in the dystrophic mouse. This study has helped to focus attention on the role of the Schwann cell as the defective cell responsible for the BL defect of murine dystrophy. When normal mouse Schwann cells were placed in contact with dystrophic mouse neurites, the Schwann cells recognized the neurites and proceeded through the proliferative-migratory stage to the ensheathmcnt stage. The final result was the synthesis of a mature, morphologically normal BL. This strongly suggests

that components intrinsic to the dystrophic neurite directing the Schwann cell to establish and maintain a permanent relationship and secrete a BL are intact. On the other hand, the inability of the dystrophic Schwann cells to form a morphologically normal BL suggests either: (1) a failure to recognize the signal from the neurites (normal or dystrophic) to begin ensheathment and BL formation ; or (2) an inability to translate this signal(s) into metabolically relevant actions. The fact that a partial, albeit incomplete BL is formed favors the sccond possibility. The results suggest that the Schwann cell rather than the neuron contains the genetic defect(s) which disallowed the complete metabolic formation of a BL. It is difficult to distinguish between the role of secreted components (from any cell type; N, S, Fb) and the influences of extracellular matrix components on the ability of the Schwann cell to function. Neurons 44, Schwann cells is and Fb 27 are all known to secrete components which may in turn influence BL formation. From these studies, it is most likely that neuronal secretion is inconsequential since the N ( + ) in the S(dy) × N(q-) cultures did not correct dy Schwann cell BL formation. These experiments also demonstrate that products secreted by normal Schwann cells did not correct the deficit in BL formation associated with the dystrophic SN explants when they were houscd in the same dish. One cannot exclude the possibility that dilution of the said secreted components occurred during the thrice weekly medium exchanges. It is interesting to note that the addition of normal Fb corrected the basal lamina defect of the dystrophic Schwann cell in our culture system. Molecules secreted by the normal Fb may substitute for those improperly formed by the dystrophic S in culture. It is known that the Fb secretes collagens, in addition to other extraccllular matrix components 27. The actuality that the post-translational modification of the collagen molecules takes place extracellularly z7 lends credence to the possibility that the same molecules necessary for enzymatic modification of Fb collagen might substitute for those which are abnormal in the dystrophic S. We have demonstrated that the Schwann cell alone is responsible for the BL abnormalities that develop during the differentiation of the dystrophic

66 P N S tissue c u l t u r e a n d t h a t the a d d i t i o n o f n o r m a l

ACKNOWLEDGEMENTS

m o u s e f i b r o b l a s t s to d y s t r o p h i c S N c u l t u r e s a p p e a r s to c o r r e c t the m o r p h o l o g i c a l a b n o r m a l i t i e s in the

T h e a u t h o r s wish to t h a n k Ms. A r t r e e J a m e s and

d y s t r o p h i c BL. T a k e n t o g e t h e r these o b s e r v a t i o n s

Ms. Lisa W a r t e l s for their i n v a l u a b l e t e c h n i c a l assis-

suggest t h a t n o r m a l d e v e l o p m e n t o f the P N S is a

t a n c e ; V i n c e n t A r g i r o for assistance with the m o r -

c o o p e r a t i v e effort b e t w e e n the n e u r o n , S c h w a n n cell

p h o m e t r i c a n a l y s i s : Dr. P a t r i c k W o o d for the c o n -

a n d the fibroblast. It m a y be necessary to define the

t r i b u t i o n o f Fig. 2; M a r k D a v i s a n d Bob F r e u n d for

role o f e a c h o f these cell types in o r d e r to fully

p r e p a r a t i o n o f the p h o t o g r a p h s and Susan M a n t i a

u n d e r s t a n d the n e u r o g e n i c a n o m a l i e s seen in m u r i n e

tbr secretarial assistance. T h i s w o r k was s u p p o r t e d

muscular dystrophy.

by a p o s t d o c t o r a l

f e l l o w s h i p f r o m the M u s c u l a r

D y s t r o p h y A s s o c i a t i o n to C . J . C . , N I H G r a n t N S 09923 to R.P.B., and G r a n t G M 2 8 0 0 2 .

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